Imaging Staphylococcus aureus biofilms in vivo reveals neutrophil exclusion by PNAG exopolysaccharide

Abstract

ABSTRACT Staphylococcus aureus forms biofilms on biotic and abiotic surfaces, enabling persistent infections that evade immune clearance. Although many S. aureus strains can produce the biofilm-associated exopolysaccharide, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), they often form protein-dominated biofilm matrices in vitro, leaving a role for PNAG unclear. Using intravital imaging in a foreign-body infection model, we found that PNAG in biofilms hinders neutrophil access and delays bacterial clearance. Neutrophil elastase was crucial for eventual biofilm clearance. In vivo PNAG labeling revealed that the exopolysaccharide forms a physical barrier that prevents neutrophils from reaching bacterial clusters. In contrast, PNAG-deficient strains permitted greater neutrophil infiltration and were cleared more rapidly than wild-type bacteria. Enzymatic degradation of PNAG with the glycoside hydrolases PgaB or dispersin B (DspB) disrupted the biofilm, restored neutrophil access, and enhanced bacterial clearance. Together, these findings identify PNAG as a key structural barrier protecting S. aureus from innate immunity and suggest that targeting PNAG with glycoside hydrolases may offer a promising therapeutic strategy for biofilm-associated S. aureus infections. IMPORTANCE The biofilm-associated exopolysaccharide PNAG is frequently expressed in Staphylococcus aureus clinical isolates but is often reduced during laboratory passage, with expression highly dependent on growth conditions. While in vitro analyses have revealed that PNAG is not a dominant matrix component, our intravital imaging of community-acquired methicillin-resistant S. aureus (CA-MRSA) skin infections demonstrates that PNAG is robustly produced in vivo and plays a central role in immune evasion. These findings highlight how PNAG function in tissue environments may be non-obvious in vitro and underscore the need for in vivo models to understand biofilm pathogenesis. By revealing PNAG as a key barrier to neutrophil-mediated clearance, this work positions PNAG and PNAG-targeting glycoside hydrolases as compelling therapeutic candidates for treating antibiotic-resistant S. aureus biofilm infections, a major cause of morbidity in both healthcare and community settings. The biofilm-associated exopolysaccharide PNAG is frequently expressed in Staphylococcus aureus clinical isolates but is often reduced during laboratory passage, with expression highly dependent on growth conditions. While in vitro analyses have revealed that PNAG is not a dominant matrix component, our intravital imaging of community-acquired methicillin-resistant S. aureus (CA-MRSA) skin infections demonstrates that PNAG is robustly produced in vivo and plays a central role in immune evasion. These findings highlight how PNAG function in tissue environments may be non-obvious in vitro and underscore the need for in vivo models to understand biofilm pathogenesis. By revealing PNAG as a key barrier to neutrophil-mediated clearance, this work positions PNAG and PNAG-targeting glycoside hydrolases as compelling therapeutic candidates for treating antibiotic-resistant S. aureus biofilm infections, a major cause of morbidity in both healthcare and community settings.